The present invention relates to a method for recovering intracellular components of materials of a biological origin, such as yeasts, algae, bacteria and/or mildews.
It is known in the art that suspensions of cellular microorganisms can be lysed by perturbation and breakage of the cell membrane for example by variations in the osmotic pressure. The simple lowering of the ionic force of the liquid environment, for example, or the addition of a surfactant with moderate stirring of the suspension, allows the cells to be swollen and allowing them to be lysed. In other cases, on the other hand, a vigorous stirring of the suspension is necessary, together with a strong thermal, mechanical or sonic action for disrupting the cell membranes, with an increase in the costs of the cell lysis. The cells of bacteria, yeasts and algae, for example, are much more resistant and their lysis for recovering the intracellular components is difficult so that resort must be made to more effective methods than osmosis for obtaining them.
In “Disruption of microbial cells for intracellular products”, Enzim Microb Tech., 1986, vol. 8, the main methods are outlined for the disruption of the cell wall, which are:
a) Mechanical methods:                by means of frictional forces between cells and solid material, for example with bead mills or X-presses or Hughes presses;        by means of frictional forces in a suspension of cells, for example with ultrasounds or with high-pressure homogenizers or with so-called French-presses.        
b) Non-mechanical methods:                by means of enzymatic, chemical lysis (with detergents, solvents or antibiotics) or physical lysis (osmotic shock or pressure).        
In industrial processes which, at the end of the fermentation, provide for the collection of micro-organisms and their cell breakage or lysis in order to release the intracellular product, an oily and aqueous enzymatic phase often accompanies the residues of the cell.
An approach to the extraction of oils from seeds followed by industry is the use of process units (malaxation) having an adequate volume stirred by helicoidal blades which rotate at a rate of 20-30 rpm and guarantee an adequate residence time (1-2 or more hours) for the disruption process for releasing the oily vacuoles. This process is carried out at a low temperature (20-30° C.) in air.
An industrial plant can generally comprise more kneaders arranged in series (in this case often superimposed in order to limit the encumbrance space) or in parallel, mechanically charged, by means of a hydraulic system, with the oil paste leaving the crusher. This process produces a bland thermal lysis which, associated with a slow remixing of the paste, allows the oily phase to be released, which is then separated by floatation process. This technology is effective for micro-organisms having dimensions larger than a few tens of microns, but it is not effective for resistant micro-organisms such as some, but not only, of the Lypomyces, Rhodotorula and Cryptococcus species. The technical parameters indicated for this process are the temperature and duration of the operation. The temperature is fundamental for the yield in the subsequent extraction and is strictly associated with the releasing phase of the cell content. The time parameter is correlated with the operative mode.
Another type of industrial machine capable of effecting this process for cases of cell lysis which require drastic temperature and pressure conditions, is a machine consisting of a fixed or rotating cylinder, equipped with internal blades or mixers capable of mixing, breaking and accompanying the cells to be lysed. This machine is used in the food industry for thermal hydrolysis and, at times in acid environments for sulfuric acids (see for example Anco-Eaglin Inc machines).
American patent application US 2010/0006515 A1 describes a machine for thermomechanical cell lysis which acts as a screw press for pressures greater than bar and temperatures higher than 120° C. In the description, it is indicated that this machine can also carry out a separation of the cellular liquids during the thermal lysing process for cellular products with membranes characterized by a strong thermal/mechanical breaking strength.
The use of glass or ceramic ball mills allows a vigorous shaking and breakage of the cells in the paste to be lysed. The method has been used for years and is considered as being applicable to the biological group of yeasts and fungi, microbacteria, spores and micro-algae. It has more recently been applied to samples of soil and small samples of vegetables and animal tissue.
Although both methods, those based on mills and on screw drums, are effective for certain types of cells, they are characterized by a high operating cost and also cost of the equipment. The latter derives from the times necessary for effecting the lysing action and, at times, due to the chemical aggressiveness of the growth environments which accompany the cellular micro-organisms, as in the case of algae, where the presence of chlorine requires the use of highly resistant materials such as alloys mainly based on nickel and chromium.
More recently, methods based on sonic or magnetic lysis processes have been used, which imply a lower final energy cost.
The intense sonication of liquids, for example, generates sound waves which propagate in liquid mediums with alternating high- and low-pressure cycles. During the low pressure cycle, a high intensity of small-sized vacuum bubbles is created in the liquid. A cavitation process is therefore used for the implosion of the liquid bubbles, which thus allows shear forces to be generated which are capable of mechanically breaking the cell structure. This effect is used for the extraction of lipids from algae. In order for the separation of the oily/lipid phase from the aqueous phase and cell debris to be effective, the addition of a solvent is required, as otherwise the cell debris would also remain englobed in the oily/lipid phase.
Other methods comprise the crushing of cells by means of high-pressure passage (20,000-30,000 psi or 140-210 MPa) through a valve having a narrow orifice. The fluid is therefore subjected to high shear forces with a consequent breakage of the cells. The shear force for optimizing the breakage of the cell is defined by controlling the pressure. The system requires high energies and a cooling for samples that require multiple steps through the system.
Mechanical homogenization is included in these mechanical methods, which operates under high-pressure conditions and with the use of a high energy density which is based on the use of pressure and microvalves for dividing the particles until they have been reduced to minimum dimensions (below a micron). The homogenizing valves are dimensioned for obtaining the desired micronization and dispersion degree at the lowest possible pressure, depending on the various applications. They are continuous industrial machines which can also manage voluminous flow-rates with low specific energies of use (for example, a machine which handles liquid flow-rates of 6 m3/h, with overpressures of 1,500 bar, requires a power equal to 315 kW).
Another approach for thermal lysis is described in literature under the name of “Hydrothermal treatment” which is experimentally applied for the lysis of algae, and is proposed for the extraction of lipids contained in oily seeds. The process is provided with experimental data for the release of the lipids contained in algae. The operation consists of a thermal lysis mechanism on a liquid-solid suspension under pressure (200 atm) and at a temperature ranging from 250 to 300° C. The final extraction of the lipids from the solid lysed cell mass, is carried out with n-hexane. The technological approach to the problem developed by this latter technology, as also those using pressurized homogenization systems, require costly industrial applications, above all for the materials used which must guarantee due resistance to the physico-chemical system composed of the suspension to be treated under the conditions of the lysing method (“Hydrothermal processing of high-lipid biomass to fuels” Johnson, Michael C., Ph. D. Massachusetts Institute of Technology. Dept. of Chemical Engineering Massachusetts Institute of Technology Issue Date: 2012).
International patent application WO 99/15638 relates to a method for modifying the structure of lipid membranes such as double lipid layers of micro-organisms, eukaryotes or prokaryotes, liposomes, vesicles or living cells. In particular, this method creates an instability (perturbation) of the lipid structure which allows the fusion of the lipid membranes without lysing the cells involved.
The method described in WO 99/15638 provides for the use of a low-temperature spray dryer) for atomizing into small drops a liquid medium such as, for example, a physiological solution, milk, blood, serum, fermentation broths and water, containing the lipid structure, which are introduced at high velocity into an environment containing vapour at a controlled temperature.
In the method of WO 99/15638, the liquid medium used for suspending the cells or lipid structures to be fused during the nebulisation, favours the fusion process of the cell membranes by lysis on the part of these. The liquid medium used must consequently not favour the breakage of these membranes but their aggregation. The objective of the process of WO 99/15638 is not to destroy the membranes of two or more different cell entities, but to keep the characteristics of the original cells unaltered, thus allowing a partial fusion of the cell membranes. In this way, the method produces molecular aggregates which combine the characteristics of the aggregated cells in the multicellular mixture.
In the lysis of a cell, one wants to recover the product of the process from micro-organisms subjected to fermentation or cultivation, and a lysing or breakage must firstly be effected, for example thermally, in a process volume, or reactor, suitable for obtaining temperatures higher than 120° C., at a pressure of at least 5 bar, for a time ranging from 10 minutes to 4-5 hours under moderate stirring, i.e. the conditions necessary for the lysis of the micro-organisms. It is subsequently, necessary to: start the reactor cooling; to extract the mass from the reactor as a suspension of solid residues, oils, water and other intracellular products; and finally an organic, or inorganic solvent must be added, or mixtures thereof, in order to obtain, with subsequent operations of solubilisation and separation of the liquids from the solids, the cell content produced of the fermentation or cultivation process.
This method is characterized in that over 90% of the energy requirements of the overall extraction process of the product of the fermentation process is due to the thermal lysis effected on the fermented micro-organisms.